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VOLATILE anesthetics interact with various cellular systems. Whereas anesthetic-lipid interactions were studied in detail in past decades, more recent emphasis has focused on the interactions between anesthetics and membrane proteins. It has indeed been shown that such interactions exist and that these modulations may be important in bringing about the anesthetic state. Ligand-gated ion channels, such as gamma-aminobutyric acid [1] receptors, have received particular attention. However, interactions with other proteins also may be important, either in modulatory roles or by inducing anesthetic side effects.

The large superfamily of G protein-coupled membrane receptors is a group of proteins that has been studied in less detail, despite the fact that it contains many members of great relevance to anesthesiologists (such as muscarinic acetylcholine and adrenergic, opiate, and eicosanoid receptors). In addition, investigations of serotonin, muscarinic acetylcholine, [2] and lysophosphatidate [3] receptors have shown interactions between anesthetics and G protein-coupled receptors.

Receptors for lipid mediators are of particular interest in this regard, because a hydrophobic ligand binding domain might be a likely site of anesthetic action. Lipid mediators-such as prostanoids, leukotrienes, and platelet activating factor-are important intercellular messengers and have pronounced biologic effects (platelet aggregation, smooth muscle contraction, pain, and inflammation). These effects are induced by activating specific membrane receptors, resulting in increased intracellular Ca2+ concentrations and changes in other second messenger systems.

Thromboxane A2(TXA2) is a prominent member of the prostanoid family and has various actions on cell and tissue functions of particular interest to anesthesiologists. For example, activation of TXA2receptors induces platelet aggregation and vascular and bronchiolar smooth muscle contraction. [4,5] Because several anesthetics exhibit effects opposite to these, we hypothesized that TXA sub 2 receptor signaling could be a target for volatile anesthetics. If anesthetics indeed inhibit signaling by hydrophobic interactions at the ligand-binding pocket, we would expect that various anesthetics would have similar effects, with potencies related to their lipid solubility. The effects of halothane, isoflurane, and sevoflurane on TXA2-induced platelet aggregation have been studied, [5,6] but results are contradictory and direct anesthetic effects on receptor functioning have not been investigated.

Xenopus oocytes form a flexible system to study recombinantly expressed G protein-coupled receptors and the influence of volatile anesthetics on their functioning. [7] Therefore, we expressed rat TXA sub 2 receptors in Xenopus oocytes and investigated the influence of halothane, isoflurane, and sevoflurane on their functioning and on the intracellular signaling pathways. The study was designed to answer the following questions:

2. Are the anesthetic effects localized at the membrane receptor or within the intracellular signaling pathway?

3. Are there differences in effect between the anesthetics that could be related to different sites of action?

Materials and Methods

Animals

The study protocol was approved by the Animal Research Committee at the University of Virginia. Female Xenopus laevis toads were obtained from Xenopus I (Ann Arbor, MI), housed in an established frog colony, and fed regular frog brittle twice weekly. To remove the oocytes, a frog was anesthetized by immersion in 0.2% 3-amino-benzoic-methyl-ester until it was unresponsive to a painful stimulus (toe pinching). Animals were operated while positioned on ice. A 1-cm-long abdominal incision was made and a lobule of ovarian tissue, containing approximately 200 oocytes, was removed and placed in modified Barth's solution (containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.41 mM CaCl2, 0.82 mM MgSO4, 0.3 mM Ca2NO3, 0.1 mM gentamicin, and 15 mM HEPES, pH adjusted to 7.6). The wound was closed in two layers and the frog was allowed to recover from anesthesia. The oocytes were defolliculated by gentle shaking in a 1 mg/ml solution of collagenase type Ia in calcium-free OR2 solution (containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH adjusted to 7.5) for 2 h. After this process, the oocytes were returned to modified Barth's solution. Microscopic observation confirmed the absence of follicle cells.

mRNA Synthesis and Injection

The rat TXA2receptor clone was obtained from Dr. K. R. Lynch (Department of Pharmacology, University of Virginia, Charlottesville, VA) as a cDNA encoding a 343 amino acid protein in the pcDNAI vector (Invitrogen, San Diego, CA). The construct was linearized with the nuclease Xho I and transcribed in the presence of capping analog by T7 polymerase, using a commercial RNA preparation kit (mMessage mMachine; Ambion, Austin, TX). Oocytes were injected with 5 ng mRNA in 30.6 nl sterile water, using an automated microinjector (Nanoject; Drummond Scientific, Broomall, PA). Microinjector functioning was confirmed by noting a slight increase in cell size during injection. The cells were then cultured in modified Barth's solution for 72 h before study.

Electrophysiologic Recording

A single defolliculated oocyte was placed in a continuous-flow recording chamber (0.5 ml volume), perfused (3 ml/min) with Tyrode's solution (containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 10 mM dextrose, 10 mM HEPES, pH adjusted to 7.4). Microelectrodes were pulled in one stage from capillary glass (BBL with fiber; World Precision Instruments, Sarasota, FL) on a micropipette puller (model 700 C; David Kopf Instruments, Tujunga, CA). Electrode tips were broken to a diameter of approximately 10 micro meter, providing a resistance of 1–3 M Omega, and filled with 3 M KCl. The cell was voltage clamped using a two-microelectrode oocyte voltage-clamp amplifier (OC725A; Warner Corp., New Haven, CT), connected to an IBM-compatible computer for data acquisition (DAS-8A/D conversion board: Keithley-Metrabyte, Traunton, MA) and analysis (OoClamp software [8]). All measurements were performed at a holding potential of -70 mV. Only cells exhibiting stable holding currents < 1 micro A during a 1-min equilibration period were included in the analysis. Membrane current was sampled at 125 Hz and recorded for 5 s before and 55 s after administration of the agonist, which allowed sufficient time for currents to return to baseline levels. Agonist (the TXA2mimetic U-46619) was delivered as a 30-micro liter aliquot during a period of 1–2 s using a hand-held micropipettor positioned approximately 3 mm from the oocyte. Agonist binding to TXA2receptors activates one or more heterotrimeric G proteins, which in turn modulate the enzyme-regulated synthesis of second messengers (Figure 1(A)). Specifically, the Gqprotein activates phospholipase C-beta (PLC-beta), which cleaves membrane phosphatidylinositolbisphosphate to inositol-1–4-5-trisphosphate (IP3) and diacylglycerol. The IP3activates receptor-channel complexes on intracellular Ca2+ stores, resulting in an increase in intracellular Ca2+ concentration. In the Xenopus oocyte, intracellular Ca2+ opens endogenous membrane Cl sup - channels, resulting in a Cl sup - flux (ICl(Ca)) measured conveniently using the voltage-clamp technique (Figure 1). Responses were quantified by integrating the current trace (Figure 1(B)) and are reported in microcoulombs (micro C). All experiments were performed at room temperature.

To study IP3- and guanosine 5'-0-(2thiodiphosphate)(GTP gamma S)-induced ICl(Ca), a third micropipette was inserted into the voltage-clamped oocyte. Tips of intracellular micropipettes were beveled with a microgrinder (Narishige EG-6 Glass Electrode microgrinder; Narishige Instrument Laboratories, Tokyo, Japan). The pipette was connected to an automated microinjector (Nanoject, Drummond Scientific). Under voltage clamp, 30 nl of 2 mM IP3or 100 mM GTP gamma S was injected, thereby activating the signaling pathway at the IP sub 3 receptor or the G protein, respectively (Figure 1(A)). Because the volume of an average Xenopus oocyte is approximately 500 nl, the injected volume approximated 5% of the oocyte volume. Therefore the estimated final concentrations were IP3100 micro Meter or GTP gamma S 5 mM. These concentrations were chosen to result in ICl(Ca) similar in size to those induced by TXA2agonist at its EC50. Induced ICl(Ca) were recorded 5 s before and 55 s after intracellular injection and analyzed as described before.

To determine the effects of halothane, isoflurane, and sevoflurane on ICl(Ca) induced by U-46619 or intracellular mediators, anesthetic was bubbled through a reservoir filled with 40 ml Tyrode's solution for at least 10 min. Air at a flow rate of 500 ml/min was used as the carrier gas. After equilibration, the solution was perfused through the recording chamber, superfusing the oocyte at a flow rate of approximately 3 ml/min; measurements were obtained after 10 bath volumes had been exchanged. Anesthetic concentrations in the recording chamber were quantified by gas chromatography (Aerograph 940; Varian Analytical instruments, Walnut Creek, CA). Results were converted to concentration in liquid using partition coefficients in Tyrode's solution at 22 [degree sign] Celsius (halothane lambda = 1.31, isoflurane lambda = 1.08, sevoflurane lambda = 0.39), and to corresponding partial pressure (vol%) at room temperature. [9,10] Aqueous concentrations equivalent to 1 minimum alveolar concentration anesthetic in air were 0.43 mM for halothane, 0.44 mM for isoflurane, and 0.44 mM for sevoflurane. Although the halothane minimum alveolar concentration-equivalent concentration is approximately twice as high as that published by Franks and Lieb, [11] our values are similar to those reported by other investigators. [2,3,6,12] Each oocyte was exposed to a single concentration of anesthetic only.

Data Analysis

Results are reported as means +/- SEM. Differences among treatment groups were analyzed using the Student's t test or the Mann-Whitney U test. If multiple comparisons were made, data were analyzed using analysis of variance followed by a t test corrected for multiple comparisons (Bonferroni). P < 0.05 was considered significant. Concentration-response curves were fit to the following logistic function, derived from the Hill equation:Equation 1where ymaxand yminare the maximum and minimum responses obtained, respectively; n is the Hill coefficient; and X50is the concentration at which the half-maximal response occurs (EC50for agonist, IC50for anesthetics).

Materials

The TXA2receptor agonist U-46619 (5-heptenoic acid, 7[[6-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]-hept-5-yl]) was obtained from Cayman Chemical (Ann Arbor, MI) and diluted in 0.1% fatty acid-free bovine serum albumin (ICN Pharmaceuticals, Costa Mesa, CA) in Tyrode's solution to appropriate concentrations. The TXA2receptor antagonist Bay U 3405 ((3R)-3-(4-fluorophenylsulfonamido)-1,2,3,4 tetrahydro [4 alpha,4b-3H]-carbazolepropanoic acid) was a gift from Bayer AG (Wuppertal, Germany) and was dissolved in the same manner. Halothane was from Halocarbon Laboratories (River Edge, NJ), sevoflurane was from Abbott International (Abbott Park, IL), and isoflurane was from Ohmeda (Liberty Corner, NJ). All other chemicals were from Sigma Chemical Company (St. Louis, MO).

Oocytes injected with 5 ng mRNA encoding the TXA2receptor responded to U-46619 with transient inward currents. The current developed after a delay of 3–5 s and consisted of a fast inward component followed by a fluctuating relaxation over several seconds (Figure 1(B)). These responses are typical for ICl(Ca) induced by Ca2+-signaling G protein-coupled receptors expressed in Xenopus oocytes. [3] The responses were concentration dependent (Figure 1(C)). Curve fitting using the Hill equation revealed a half-maximal effect concentration (EC50) of 3.2 x 10 sup -7 +/- 1.1 x 10 sup -7 M and a Hill coefficient of 0.8 +/- 0.2.

I sub Cl(Ca) Induced by U-46619 Is Mediated by TXA sub 2 Receptors

To confirm that the ICl(Ca) induced by U-46619 is indeed mediated by recombinantly expressed TXA2receptors, we applied the agonist (1) to uninjected cells to determine if endogenous TXA2receptors were present and (2) to cells expressing the TXA2receptor, 1–2 min after application of the specific TXA2receptor antagonist Bay U 3405 (10 sup -5 M). U-46619 (10 sup -4 M)(had no effect in uninjected cells (data not shown). Treatment with Bay U 3405 suppressed the responses to U-46619 (10 sup -6 M) to 15% of control (Figure 2). Together these findings confirm that the agonist indeed signaled through recombinantly expressed TXA2receptors.

After confirming that the receptor was functional, that the agonist was acting selectively on expressed receptors, and that the intracellular signaling pathway was as described for this receptor (Figure 1(A)), we tested the ability of halothane to interfere with TXA2signaling. Halothane at clinically relevant concentrations inhibited ICl(Ca) induced by U-46619 (10 sup -6 M)(Figure 4(A)). Curve fitting using the Hill equation revealed a half-maximal inhibitory concentration (IC50) of 0.47 +/- 0.05 mM (0.83%) and a Hill coefficient of 1.6 +/- 0.2 (Figure 4(B)). The highest halothane concentration tested, 1.07 mM (2.5%), suppressed signaling to 17% of control. To determine if the effect of halothane was reversible, we studied U-46619-induced currents under three conditions: in the absence and presence of halothane and after washout of halothane with anesthetic-free solution (Figure 4(C)). All three measurements were performed in the same oocyte, separated by washes of at least 10 min. A high concentration of halothane (1.07 mM, 2.5%) was used because it was considered more likely to cause irreversible effects. Although U-46619-induced responses were depressed more than 80% in the presence of anesthetic, ICl(Ca) obtained in response to U-46619 after wash with anesthetic-free solution were similar in size to control responses (Figure 4(C)), indicating that halothane's effect is reversible.

Halothane Acts in a Competitive, Isoflurane in a Noncompetitive Manner

If anesthetic inhibition of TXA2signaling results from hydrophobic interactions at the ligand-binding pocket, we would expect that the effect would be competitive (that is, fully reversible by high agonist concentrations) in nature. In contrast, a site of action elsewhere in the signaling pathway would likely be noncompetitive (that is, not fully reversible by high agonist concentrations). To determine if halothane and isoflurane act as competitive or noncompetitive antagonists on TXA2signaling, the concentration-response relation for U-46619 was determined in the presence of halothane 0.56 mM (1%, close to halothane's IC50) or isoflurane 0.7 mM (1.75%, close to isoflurane's IC50). Halothane acted in a competitive manner: it did not affect Emaxbut caused a parallel shift of the concentration-response relation to the right. This increased the U-46619 EC50by approximately two orders of magnitude to 1.1 x 10 sup -5 M (Table 1). In contrast, isoflurane acted in a noncompetitive manner: It did not affect the U-46619 EC50(5.3 x 10 sup -7 M) but decreased Emaxto 70% of control (Table 1, Figure 6).

We also tested the ability of sevoflurane to inhibit TXA2signaling. In contrast to the other anesthetics tested, sevoflurane, at concentrations up to 5%(0–1.3 mM, 2.92 minimum alveolar concentration), [14] did not affect TXA2signaling (Figure 7).

Halothane, Isoflurane, and Sevoflurane Have No Effect on Intracellular Signaling Pathways

To determine the site of action of halothane on the TXA2signaling pathway, we activated segments of the intracellular pathway directly by intracellular microinjection of second messengers. IP3directly activates its receptor-channel on intracellular Ca2+ stores; GTP gamma S irreversibly activates G proteins (Figure 1(A)). Therefore, a lack of anesthetic effects on ICl(Ca) induced by these mediators would indicate the receptor or the coupling between G protein and receptor as a site of anesthetic action. IP3and GTP gamma S induced ICl(Ca) in uninjected oocytes. Average responses were 3.9 +/- 0.2 micro C for IP3-induced ICl(Ca) and 4.4 +/- 0.4 micro C for GTP gamma S-induced ICl(Ca). Neither halothane, isoflurane, nor sevoflurane affected these currents: IP3-induced responses in the presence of 1.07 mM halothane (2.5%), 1.32 mM isoflurane (3.3%), or 1.3 mM sevoflurane (5%) were 101 +/- 19.6%, 89 +/- 22.4%, and 93 +/- 14.07% of control, respectively. GTP gamma S-induced responses were 131 +/- 25.4% of control in the presence of halothane, 122 +/- 18.6% in the presence of isoflurane, and 120 +/- 32.5% of control in the presence of sevoflurane (not significantly different from control;Figure 8). When 10-fold greater or 10-fold smaller concentrations of IP3and GTP gamma S were injected, the anesthetics were similarly without effect (data not shown).

Figure 8. The intracellular pathway is not modulated by volatile anesthetics. (A) ICl(Ca) induced by intracellular microinjection of GTP gamma S are not inhibited by sevoflurane, isoflurane, or halothane at concentrations of 1.3 mM, 1.3 mM, and 1.07 mM, respectively. Intracellular injection of water does not induce ICl(Ca). (B) Intracellular microinjection of the second messenger inositol 1–4-5 trisphosphate (IP3) induces ICl(Ca). Currents are not inhibited by halothane (1.07 mM), isoflurane (1.32 mM), or sevoflurane (1.3 mM).

Thromboxane A2is a potent stimulator of platelet aggregation [5,6,15] and a constrictor of vascular [5,16] and respiratory smooth muscle. [17] Its function is counterbalanced with that of prostacyclin, which inhibits platelet aggregation and elicits vasorelaxation. Disruption of this balance in favor of TXA2has been suggested to play a role in thrombosis, asthma, and unstable angina, as well as in myocardial infarction. [18] Thus TXA2receptor antagonists are of considerable therapeutic importance. [17] Therefore acute inhibitory effects of anesthetics on TXA2signaling may be beneficial; in addition, short-term modulation of TXA2signaling during anesthesia may result in long-term beneficial effects as well. [19]

Hirakata et al. [20] determined the amino acid sequence of the human TXA2receptor by cloning it from a human placental cDNA library. Nilsing et al. [12] recently cloned the gene for the human TXA2receptor and found no evidence for additional genes. Thus only one TXA2receptor type appears to exist. This is unusual, because for most other members of the G protein-coupled receptor superfamily the existence of several receptor subtypes has been demonstrated. The present study had the advantage that our findings are not subtype dependent.

Because of these inconsistencies, and because functioning of the TXA2receptor per se had not been studied, we did this investigation. We found no interference of sevoflurane with TXA2signaling, in agreement with the lack of receptor binding observed by Hirakata et al. [6] Halothane did inhibit receptor functioning in a competitive manner, consistent with its effects on receptor binding (although the functional effects are notable at much lower concentrations). The noncompetitive interaction with receptor functioning observed for isoflurane is consistent with its lack of effect on receptor binding. However, the lack of effect of isoflurane on platelet aggregation as reported by Hirakata et al. [6] cannot be reconciled with either Blaise et al.'s [5] or our own data. Although the special properties of our model (such as use of room temperature, amphibian membrane, and G protein) should be kept in mind, previous studies show that the anesthetic effects on receptors expressed in Xenopus oocytes are similar to those observed in other models. [2,3,21,22] In addition, our data are consistent with those of Blaise et al. [5]

Site of Action

Our experiments with microinjected intermediates show that the intracellular signaling pathway is unaffected by the anesthetics. Therefore, the receptor itself is the most likely site of action. Because halothane's effect is competitive and it interferes with ligand binding, [6] its action is probably at the ligand-binding site. Because halothane is the most lipid soluble of the three anesthetics tested (oil-water partition coefficient 310 [23]), we hypothesize that the site of halothane's action is hydrophobic. Yamamoto et al. [15] modeled the structure of the TXA2receptor and identified several amino acid residues likely involved in ligand binding, as well as a large hydrophobic pocket among these amino acids. Based on this combination of data, we postulate that halothane interacts with this hydrophobic pocket.

In contrast, inhibition by isoflurane is noncompetitive, and the anesthetic does not interfere with ligand binding. [6] Therefore its (allosteric) site of action is unlikely to be at the ligand-binding pocket. Its lipophilicity (oil-water partition coefficient 170 [23]) is less than that of halothane, making it a less suitable candidate for interaction with the lipophilic pocket. In agreement, sevoflurane (oil-water partition coefficient 32 [23]) was completely without effect. The exact site of action of isoflurane cannot be determined from the present studies. However, mutation analysis might make localization of this site possible.

These findings reinforce the concept that different anesthetics may have different sites of action within the same molecular structure. In other words, even if anesthetics have similar actions on a molecule, it does not follow that they act on the same site. Eckenhoff [24] recently demonstrated the existence of multiple binding domains for inhalational anesthetics in the nicotinic acetylcholine receptor. Similarly, different binding domains may exist within the gamma-aminobutyric acidAreceptor complex. Halothane and isoflurane enhance the ligand binding of alpha1gamma2gamma-aminobutyric acidAreceptors. However, cotransfection with the beta2subunit reduced the efficacy of both isoflurane and halothane, whereas cotransfection with the beta3subunit increased the efficacy of isoflurane but not halothane. [25] Thus at least several signaling molecules may have multiple, separate sites of action for volatile anesthetics.

Conclusions

Whereas halothane and isoflurane inhibit TXA2receptor functioning, halothane acts in a competitive and isoflurane acts in a noncompetitive manner. In contrast, sevoflurane has no effect. The site of action appears to be the receptor molecule itself. The site of halothane's action is most likely the hydrophobic pocket in the ligand-binding domain. In contrast, isoflurane most likely acts at an allosteric site.